Dengue in Jeddah, Saudi Arabia,

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1 by Mazen Fakeeh and Ali M Zaki Virus Laboratory, Dr Solimon Fakeeh Hospital, Jeddah, Saudi Arabia Abstract Dengue virus (DEN-2) was first isolated from a fatal case of dengue haemorrhagic fever (DHF) in an adult in Jeddah, Saudi Arabia, in A surveillance system for dengue was established in 1994 and since then clinical, virological, serological and RT-PCR techniques were used to confirm dengue cases and, as a guide, to implement vector control measures in risk areas. During February 1994-December 2002, a total of 1,020 suspected clinical cases were examined by laboratory methods. Dengue virus infection was confirmed in 319 (31.3%) cases, 209 by virus isolation and the rest by serological techniques. DEN-2, DEN-1 and DEN-3 were detected between 1994 and 2002 in that order of frequency. Using IgG immunofluorescent assay or haemagglutination inhibition (HI) test, the prevalence of dengue reactive antibodies in the suspected group was confirmed in 515 (50.5%) of the 1,020 samples tested. The application of reverse transcriptase-polymerase chain reaction (RT-PCR) on culture-positive blood samples gave a specificity and sensitivity of 100% and allowed rapid diagnosis within one day. All cases that were diagnosed by laboratory methods as dengue had leucopenia, thrombocytopenia and elevated liver enzymes, ALT-AST. There were only two fatal cases; one of dengue haemorrhagic fever and another of dengue shock syndrome. It can be said that: (i) three dengue serotypes (DEN 1, 2 and 3) were detected in Jeddah; (ii) nearly all cases were non-complicated, 99.4% of them were of dengue fever; (iii) prevalence of dengue antibodies was in 50.5% of all suspected cases; and (iv) RT-PCR is a rapid, sensitive and effective method for diagnosis. Limited rains, active case-finding and effective anti-mosquito measures helped to bring the disease under control and were probably responsible for the very small numbers of new indigenous dengue cases after the 1994 outbreak. Keywords: Dengue, dengue fever, Jeddah, Saudi Arabia. Dengue Bulletin Vol 27,

2 Introduction Dengue fever (DF), and its more severe form known as dengue haemorrhagic fever (DHF), is the most important arthropod-transmitted viral disease of humans in the world today with one third of the world's population at risk. Epidemics of a dengue-like disease appeared in the Arabian Peninsula in the late 18 th century. The disease was described in Zanzibar, Dar es Salam, the East African coast, Arabia (Aden, Mecca, Madera), and Jeddah, in Saudi Arabia (1). In 1994, the dengue virus was isolated in Jeddah, Saudi Arabia, from a fatal case of DHF at the Dr Fakeeh Hospital (2). Active surveillance was established in 1994, based on clinical, followed by laboratory, methods to evaluate the prevalence and incidence of dengue, the serotypes detected, and the efficacy of the laboratory methods used for its diagnosis. This paper (2) reviews the surveillance data from ; and this report extends these results to 2002 and adds data obtained by reverse transcriptase-polymerase chain reaction (RT-PCR). Materials and methods Case selection All cases suspected of having dengue, with non-specific fever, with no localizing signs or symptoms, were examined clinically and blood samples collected when suspected cases were first seen. Virus isolation Blood samples were inoculated on C6/36 and after the seventh day, cells were examined for cytopathogenic effects (CPE) and also examined by indirect immunoflourescent assay (IFA) for detection of dengue antigen (3,4). Serological methods Haemagglutination-inhibition (HI) antibody method of Clarke and Casals (5) was modified for use in microtiter format. IgM capture ELISA was applied according to the method described by Kuno et al. (6). Antibodies of the IgG type were detected using IFA (7). Nucleic acid RT-PCR was done using commercial kits for the extraction of viral RNA, amplification of cdna and detection of amplified products. Viral RNA was extracted from tissue culture supernatant or serum using Qiagen RNA kit (Qiagen Germany, catalogue number 29504). Primers and probes were as described by Lanciotti (8). RT-PCR was done using Qiagen one-step RT-PCR kit (Qiagen, Germany, catalogue number ) in 50 ul volumes. Amplified products were detected, using DNA enzyme immunoassay (DEIA) (Dia Sorin Biomedica Saluggia, Italy) according to the manufacturer's instructions. Results The number of clinically suspected cases was 1,020 (738 male and 282 female). Dengue virus infection was confirmed in 319 cases (31.3%). Virus isolation was obtained in 209 cases (65.5%) out of 319 cases and IgM capture ELISA detected 110 cases (34.5%). Three dengue serotypes, DEN-2, DEN-1 and DEN-3, were detected. Most of the cases (289, 90.6%) out of 319 were diagnosed in 1994 and the rest over the next eight years (Table 1). 14 Dengue Bulletin Vol 27, 2003

3 Table 1. Distribution of suspected and confirmed dengue cases, Year Number of suspected cases Number of confirmed cases Total The application of RT-PCR gave results comparable to virus isolation in all the samples that were tested. Typing of dengue isolates by RT-PCR, followed by hybridization using DEIA method gave the same results as culture followed by the use of type-specific monoclonal antibodies in an IFA (Table 2). The prevalence of IgG antibodies by IFA assay showed 515 (50.5%) of the total tested cases (1,020) had IgG antibodies in the first sample submitted for laboratory diagnosis. IgM detected 110 cases, mostly by the end of the first week of infection. Table 2. Results of RT-PCR followed by DEIA compared to gel electrophoresis after nested PCR and IFA following virus culture Samples RT-PCR followed by DEIA Nested PCR followed by gel electro-phoresis Virus culture followed by IFA detection DEN-1 positive DEN-2 positive DEN-3 positive Control negative Dengue infected C6/36 cells Dengue noninfected C6/36 cells 28/28 28/28 28/28 40/40 40/40 40/40 12/12 12/12 12/12 0/30 0/30 00/30 20/20 20/20 20/20 00/20 00/20 00/20 DEIA = DNA enzyme immunoassay, IFA = Indirect immunofluorescent assay Dengue Bulletin Vol 27,

4 Discussion and conclusions Dengue fever made its first appearance in Jeddah in 1994; by the end of the year, 289 cases had been diagnosed. Since that time, continuous surveillance showed that dengue was no longer endemic in Jeddah. Jeddah is a central area where a large number of pilgrims visiting Mecca transit every year for Haj (visiting Islamic holy places). The Haj provides a fertile opportunity for the introduction and exchange of infectious agents among pilgrims, including dengue viruses. Pilgrims come from many countries in South-East Asia, India, Indonesia, Malaysia, and Pakistan. Some of these persons may be in the incubation period of dengue infection and may actually be viremic during the time of Haj, infecting mosquitoes and subsequently infecting other pilgrims and residents. Also, pilgrims come from Africa, so in Jeddah and Mecca, there may be an exchange of dengue viruses among pilgrims. However, the little (less than 60 mm/year) rain and dry weather most of the year does not favour efficient spread of dengue. A case-control study indicated that water storage container facilities in or near construction sites were the focal points of dengue virus transmission in Jeddah as, in spite of the low rainfall, water storage containers served as the breeding sites for Aedes aegypti, the vector species (12). It was during 1994 that two DEN-2 isolates were obtained and one of these was from a fatal haemorrhage case. Immediately after the isolation of DEN-2 virus surveillance was established by the Ministry of Health and information and guidelines were passed on to clinicians in the region. Clinicallysuspected cases based on WHO criteria were sampled and blood was tested at the Dr Solimon Fakeeh Hospital by virus isolation, IgM capture ELISA, and finally by an IFA (later RT-PCR was applied). Clinically, all cases were found to be of noncomplicated dengue fever, except for two cases that were fatal. Despite the high prevalence of dengue antibody in the examined suspected cases, among the confirmed cases only these two showed DHF or DSS. Inoculation of acute phase sera on C6/36 cells detected 209 cases, mostly in the first days of the disease, a common finding elsewhere (4,9,13,14). Most of the confirmed cases were adults between years of age and there were more male than female, a pattern consistent with the exposure at construction sites in Jeddah. The male/female ratio was similar to observations made in other dengue endemic areas in Brazil, Puerto Rico and, more recently, in the Philippines. Three dengue serotypes were detected over the nine-year period from 1994 to 2002; DEN-2 (138 cases), DEN-1 (58 cases), and DEN-3 (13 cases). There were no DEN-4 isolates. The paucity of cases of DHF in the population in Jeddah could be attributed to factors such as viral, racial or nutritional. Typing of the disease in this study was done using specific monoclonal antibodies (3,4). Most isolates were in 1994 (186) and the rest of the isolates were in the following eight years. IgM capture ELISA detected 110 cases that were culture negative, presumably because of the delay in collecting the blood samples. Samples were often taken by the end of the first week of the presentation of symptoms and signs. The IgM ELISA has a high sensitivity and specificity for dengue infection (9,10,11). Application of RT-PCR, followed by DNA enzyme immunoassay for the detection of amplified products, gave comparable results 16 Dengue Bulletin Vol 27, 2003

5 to virus isolation on tissue culture followed by typing with monoclonal antibodies. The main advantages of the RT-PCR method is its rapidity, done in one day, sensitivity and specificity of diagnosis (8). The city of holy Mecca is 75 km from Jeddah. During Haj, large numbers of pilgrims come from disease-endemic areas all over the world, with the possibility of introduction of dengue viruses. Rapid and frequent travel between Jeddah and other endemic areas of the world favours the continued introduction of other strains and serotypes. It is important to maintain dengue surveillance at high levels in order to detect early dengue activity and to take effective steps for the control of the vector. Acknowledgements We thank Dr Robert E. Shope of the University of Texas Medical Branch at Galveston for his encouragement in this work and for reviewing the manuscript. We also thank Miss Amany Ibrahim Yousef for her technical assistance. References 1. Gubler DJ. Dengue and dengue haemorrhagic fever: Its history and resurgence as a global health problem in DJ Gubler and G Kuno ed. Dengue and dengue haemorrhagic fever. CAB International, Willingford, Oxon, OX 10 8DE.UK, 1997: Fakeeh M and Zaki AM. Virologic and serologic surveillance for dengue fever in Jeddah, Saudi Arabia. The American Journal of Tropical Medicine and Hygiene, 2001, 65: Henchal EA, Mc Cown JM, Seguin MC, Gentry MK and Brandt WE: Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescent assay. American Journal of Tropical Medicine and Hygiene, 1983, 32: Gubler DJ, Kuno G, Sather GE, Velez M and Oliver A. Mosquito cell culture and specific monoclonal antibodies in surveillance for dengue viruses. American Journal of Tropical Medicine and Hygiene, 1984, 33: Clarke DH and Casals J. Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses. American Journal of Tropical Medicine and Hygiene, 1958, 7: Kuno G, Gomez I and Gubler DJ. An ELISA procedure for the diagnosis of dengue infection. Journal of Virological Methods, 1991, 33: Shope R and Sather G. Arboviruses. Lennette EH, Schmidt NJ eds Diagnostic procedures for viral, rickettsial, and chlamydial Infections. Fifth edition Washington DC: American Public Health Association, 1979: Lanciotti RS, Calisher CH, Gubler DJ and Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples using reverse transcriptasepolymerase chain reaction. J Clin Microbiology, 1992, 30: Gubler DJ and Sather G. Laboratory diagnosis of dengue and dengue haemorrhagic fever. Proceedings of the international symposium on yellow fever and dengue. Rio de Janiero, Brazil May 15-19, Innis BL, Nisalak A, Nimmannitya S, Kusalerdchariya S, Chongswadi V, Suntayakorn S, Puttisri P and Hoke CH. An enzyme linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. American Journal of Tropical Medicine and Hygiene, 1989, 40: Dengue Bulletin Vol 27,

6 11. Cardosa MJ, Tio PH, Nimmannitya S, Nisalak A and Innis B. IgM capture ELISA for detection of IgM antibodies to dengue virus: Comparison of 2 formats using hemagglutinins and cell culture derived antigens. South-East Asian Journal of Tropical Medicine and Public Health, 1992, 23: Ghaznawi HI, Al-Khateeb TO, Akbar N, Afifi H and Nasser A. Surveillance for dengue fever in Jeddah. Eastern Mediterranean Health Journal, 1997, 3: Chungue E, Burucoa C, Boutin JP, Philippon G, Laudon F, Plichart R, Barbazan P, Cardines R and Roux J. Dengue 1 epidemic in French Polynesia, : Surveillance and clinical, epidemiological, virological and serological findings in 1,752 documented clinical cases. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1992, 86: Technical Advisory Group on dengue haemorrhagic fever/dengue shock syndrome. Dengue haemorrhagic fever diagnosis, treatment and control. Geneva, Switzerland: World Health Organization, Dengue Bulletin Vol 27, 2003